Labeling of synthetic polynucleotides is typically accomplished by one of two methods. One method consists of using a derivatized solid support, such as controlled pore glass or polystyrene, where a terminal label is attached to the solid support through a covalent linkage. The desired polynucleotide is synthesized off a free hydroxyl group present on the support-bound label, and the labeled polymer is then cleaved from the solid support when a desired length is achieved. Another method requires synthesizing the polynucleotide with a reactive terminal or internal linker, such as an amino or thiol linker, and attaching a label following synthesis employing a reactive derivative such as an active ester, maleimide, or iodoacetamide.
While the above methods can be productively used to produce a variety of labeled polynucleotides, they suffer from several disadvantages. Some useful labels are not stable when repeatedly exposed to the conditions required for polynucleotide synthesis, thereby limiting the number of support-bound species that can be utilized for labeling. Use of reactive terminal linkers requires a multi-step conjugation chemistry employing an excess of (often very expensive) label and requires at least one purification step to isolate the labeled biopolymer from excess of reactants. This method is often very inefficient due to competing side-reactions that cause breakdown of the reactive dye, and to the poor solubility of many dye derivatives in aqueous solution. Additionally, linker-modified supports for introducing reactive functionalities onto the 3′-terminus of a polynucleotide often result in low yield and poor quality of the cleaved polynucleotide, further exacerbating the inefficiency of this approach.
Thus, there is a need in the art for methods and compositions for making phosphoramidites and supports that bear protected linking groups; wherein the protected linking group chemistry is compatible with, or orthogonal to, other protecting groups commonly employed in nucleic acid synthesis. Such phosphoramidites and supports provide one or more unique reactive sites on the polynucleotide that enable automated labeling of the polynucleotide following chain assembly.